Our laboratory conducts investigator-initiated research into the biology of HIV-1 using molecular biology, biochemical, and cellular biological techniques. We contribute to the goals of the NCI by developing research expertise and advancing the understanding of the virus and AIDS. Cellular proteins in HIV-1 and assembly: Our efforts to identify and characterize the host proteins inside HIV-1 virions and their relevance in the viral life-cycle were continued during this period. Cellular proteins are incorporated into retroviruses and may play important roles in HIV-1 biology and pathogenesis. Additionally these cellular proteins might reveal important insights into the interactions between HIV-1 and cellular pathways during assembly. Along with the Retroviral Pathogenesis Laboratory, we have developed a CD45 immunoaffinty depletion technology that produces highly purified virus preparations (J. Virol. in press). This approach complements our previously established expertise in removing non-viral sources of cellular proteins. Together, these techniques provide powerful tools to completely and unambiguously analyze the cellular proteins both inside and outside HIV-1 virions. Currently, we have begun to analyze these highly purified virion preparations using traditional biochemical analyses and state-of-the-art mass spectrometry sequencing. While most of our studies have focused on virus produced from relevant transformed T cell lines (Rev. Med. Virol. 12:359), HIV-1 virions produced from important primary cell types (i.e., monocyte-macrophages and T cells) are also being examined. From these studies, the relevance and roles of several cellular proteins in HIV-1 biology were intensively investigated. The participation of the ubiquitin-proteasome and the endosomal pathways in HIV-1 assembly and budding were studied (J. Biol. Chem. 277:38818). We also found the budding of other retroviruses are affected by proteasome inhibitors in ways different from HIV, suggesting a new model for virus/cell interactions during assembly (J. Virol. 77:3384). The cellular basis for the sensitivity of HIV-1 budding to proteasome inhibitors was also investigated in collaboration with Dr. Ulrich Schubert of NIAID at NIH. Additionally, collaborative studies with Markus Thali of the University of Vermont has lead to new ways to image HIV-1 in living cells, which implicate the endosomal pathway as a partner in HIV-1 budding. Together, these studies can potentially explain several unknown aspects of HIV-1 budding. In turn, understanding HIV-1 budding might identify new targets for anti-retroviral therapy. Mechanisms of HIV-1 assembly: Experiments with a series of mutants based on a nearly complete deletion of the HIV-1 NC protein from the HIV-1 Gag polyprotein demonstrated that, contrary to expectations, this protein is dispensable for virion formation (J. Virol. 77:5547). Additional data suggest, that regulation of HIV-1 protease processing is a key part of viral assembly. Particles produced by this mutant are not infectious, being <106 as infectious as wild-type. The production of noninfectious virions by this mutant makes this another potential method to produce a whole particle inactivated HIV-1 vaccine. Reversible transformation of primary cells. We have developed a system to isolate and immortalize antigen specific T cell lines from human blood. These cell lines, generated from soluble- or allo-antigen-stimulated patient peripheral blood mononuclear cells maintain their antigen responsiveness and primary cell characteristics. This system should greatly assist the analysis of the T cell component of the immune response to viral antigens as well as other medically relevant antigens. We are also developing a method to reverse the immortalization of these T cell lines by excising the immortalizing genes so these cells could be used as primary T cells, e.g., in a clinical setting for adoptive therapy. Selective and reversible T cell immortalization promises to allow for the dissection of the host immune response to retroviruses or vaccines, the development of appropriate cell lines for vaccine production and studies, and potential applications for cancer therapy.